For Peer Review
Draft Manuscript for Review
NPHS2 p.V290M mutation in late-onset steroid-resistant
nephrotic syndrome
Journal: Pediatric Nephrology
Manuscript ID: pedneph-12-07-0412.R1
Manuscript Type: Original Article
Date Submitted by the Author: 19-Oct-2012
Complete List of Authors: Kerti, Andrea; Semmelweis University, Ist Department of Pediatrics Csohány, Rózsa; Semmelweis University, Ist Department of Pediatrics Szabó, Attila; Semmelweis University, Ist Department of Pediatrics Árkossy, Ottó; FMC Szépvölgyi Dialysis Centre, Szent János Hospital, Nephrology Outpatient Dept Sallay, Péter; Semmelweis University, Ist Department of Pediatrics Moriniére, Vincent; Hôpital Necker, INSERM U983 Vega-Warner, Virginia; University of Michigan, Departments of Pediatrics and Human Genetics Nyírı, Gábor; Semmelweis University, Clinical and Research Center for Molecular Neurology Lakatos, Orsolya; University of Pécs, Department of Pediatrics Szabó, Tamás; University of Debrecen, Medical and Health Science Center, Pediatric Institute Lipska, Beata S.; Medical University of Gdansk, Department of Biology and Genetics; University of Heidelberg, Division of Pediatric Nephrology, Center for Pediatrics and Adolescent Medicine Schaefer, Franz; University of Heidelberg, Division of Pediatric Nephrology, Center for Pediatrics and Adolescent Medicine Antignac, Corinne; Hôpital Necker, INSERM U983 Reusz, George; Semmelweis University, Ist Department of Pediatrics Tulassay, Tivadar; Semmelweis University, Joint Research Laboratory of the Hungarian Academy of Sciences and the Semmelweis University Tory, Kalman; Semmelweis University, Ist Department of Pediatrics
Keywords: genetics, nephrotic syndrome, NPHS2, podocin, mutation screening, algorithm
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
For Peer Review
1
NPHS2 p.V290M mutation in late-onset steroid-resistant nephrotic syndrome
Andrea Kerti1, Rózsa Csohány1, Attila Szabó1, Ottó Árkossy2, Péter Sallay1, Vincent
Moriniére3, Virginia Vega-Warner4, Gábor Nyírı5, Orsolya Lakatos6, Tamás Szabó7,
Beata S. Lipska8,9, Franz Schaefer9, Corinne Antignac3, George Reusz1, Tivadar
Tulassay10, Kálmán Tory1
1 Ist Department of Pediatrics, Semmelweis University, Budapest, Hungary
2 FMC Szépvölgyi Dialysis Centre, Szent János Hospital, Nephrology Outpatient Dept, Budapest, Hungary
3 INSERM U983, Hôpital Necker, Paris, France
4 Departments of Pediatrics and Human Genetics, University of Michigan, Ann Arbor, Michigan, USA
5 Clinical and Research Center for Molecular Neurology, Semmelweis University
6 Department of Pediatrics, University of Pécs, Hungary
7 University of Debrecen, Medical and Health Science Center, Pediatric Institute, Debrecen, Hungary
8 Department of Biology and Genetics, Medical University of Gdansk, Poland
9 Division of Pediatric Nephrology, Center for Pediatrics and Adolescent Medicine, University of Heidelberg, Germany
10 Joint Research Laboratory of the Hungarian Academy of Sciences and the Semmelweis University, 1st Department of Pediatrics
Corresponding author:
Kálmán Tory
Cell: +36-20-8258166; Fax: +36-1-3138212
Ist Department of Pediatrics, Semmelweis University, Budapest
Bókay J. u. 53., Budapest, 1083 Hungary
Page 1 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
2
Abstract
Background. The most frequently mutated gene of steroid-resistant nephrotic
syndrome (SRNS) is NPHS2. Current guidelines propose the sequencing of all
NPHS2 exons only in childhood-onset SRNS.
Methods. A cohort of 38 Hungarian patients with childhood-onset nephrotic-range
proteinuria was screened for NPHS2 mutations. The frequency of the p.V290M
mutation in late-onset SRNS was examined in the French and PodoNet cohorts.
Results. Seven of 38 Hungarian patients carried NPHS2 mutations on both alleles.
Two of them – diagnosed with proteinuria through school-screening programs at the
age of 9.7 and 14 years – have not developed nephrotic syndrome in childhood. The
first, an 18-year-old man, homozygous for p.V290M, has never developed edema.
The second, a 31-year-old woman – compound heterozygous for p.V290M and
p.R138Q – was first detected to develop hypoalbuminemia (<30g/l) and edema at the
age of 24.3 and 27.5 years, respectively. Both patients have a normal GFR. The
mutation p.V290M was carried by 3/38 patients in the Hungarian cohort, by 2/95
patients with late-onset SRNS in the PodoNet cohort and 0/83 patients in the French
cohort.
Conclusions. We propose that not only the p.R229Q variant, but also the p.V290M
mutation should be screened in Central and Eastern European patients with a late-
onset SRNS.
Keywords: genetics, nephrotic syndrome, NPHS2, podocin, mutation screening,
algorithm
Page 2 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
3
Introduction
Nephrotic syndrome is the consequence of the glomerular filtration barrier’s damage
and refers the clinical picture of heavy proteinuria, hypalbuminemia, edema and
hyperlipidemia. The majority of patients with nephrotic syndrome respond to steroid
therapy and have a good clinical prognosis. In contrast, approximately 10% of
children and 40% of adults are steroid-resistant and progress to ESRD [1-3].
Based on its etiology, two forms of steroid-resistant nephrotic syndrome (SRNS) can
be distinguished, an immune and a genetic form. As the treatment in the two forms is
different, their differentiation is clinically important. This is primarily based on the
identification of the causative mutation in one of the ten genes identified thus far in
isolated SRNS [4-14]. Out of them, NPHS2, encoding podocin, is the most frequently
mutated gene in all but the neonatal age group [13, 15, 16].
NPHS2 mutations were first described in children presenting with SRNS before the
age of 6 years who progressed to ESRD during the first decade of life. Histologically,
minimal change disease (MCD) were found on early biopsy samples and focal
segmental glomerulosclerosis (FSGS) at later stages. [13]
Based on large cohort studies, among patients carrying NPHS2 mutations on both
alleles, the onset of SRNS depends significantly on the type of the mutation [17, 18].
An onset above the age of 10 years is exceptional and have been reported
exclusively in patients carrying a missense or a splice site mutation [17-21].
Nevertheless, even these patients have developed nephrotic syndrome till the age of
16 years, and progressed to ESRD till the age of 26, the latest [17, 22].
However, the clinical phenotype caused by NPHS2 can be even less severe due to
the variant p.R229Q [23]. This variant when found in trans-association with one
Page 3 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
4
pathogenic NPHS2 mutation, was reported in patients developing SRNS as late as
39 years of age and ESRD at the age of 43.5 years [22].
Based on these observations, sequencing of all NPHS2 exons has been proposed
only in children presenting with SRNS before the age of 14 years. In patients with a
late-onset SRNS, only the p.R229Q variant is proposed to be screened at the first
step, followed by sequencing of the other exons in case of its presence [4, 16].
Here we present a late-onset, slowly progressing SRNS in two unrelated patients
carrying the p.V290M NPHS2 mutation. Mutation-screening in these patients – based
on the current guidelines – would have failed. We propose that this missense
mutation should also be screened in Central and Eastern European patients with
late-onset SRNS, where its allele frequency is not negligible.
Page 4 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
5
Methods
Out of a cohort of 47 Hungarian unrelated patients with nephrotic-range proteinuria
(>40mg/m2/h) diagnosed before 14 years of age and unresponsive to steroid-
treatment, 7 patients were excluded for syndromic SRNS, 1 patient for a histology of
diffuse mesangial sclerosis and 1 for recurrence after renal transplantation. Thus, a
total of 38 patients with nephrotic-range proteinuria with FSGS (n=30) or MCD (n=8)
on histology have been selected for NPHS2 mutation-screening. Thirty-six of 38
patients developed nephrotic syndrome either at diagnosis (n=32) or later, but before
the age of 14 years (n=4). Eight patients were later reported to achieve remission
after cyclosporine therapy. None of the families was known to be consanguineous.
One case was familial with an affected sibling pair.
Genomic DNA was isolated from peripheral blood by standard methods, after
obtaining informed consent from the patients or their parents. The 8 exons and the
adjacent intronic junctions of NPHS2 have been directly sequenced by one of three
laboratories: (1) Ist Department of Pediatrics, Semmelweis University, Budapest,
Hungary (2) INSERM U983, Necker Hospital, Paris, France (3) University of
Michigan, Ann Arbor, Michigan, USA.
Screening of the p.V290M mutation in large SRNS cohorts
The frequency of the mutation c.868G>A (p.V290M) was examined in late-onset
SRNS in two large cohorts: (1) in 83 patients from the French cohort originating
mainly from Europe and North Africa with an onset of SRNS between the ages of 14
and 70 years (average: 29 years) and (2) in 95 patients from the PodoNet cohort
originating mainly from Italy (n=42), Turkey (n=27), Germany (n=9), and Poland (n=9)
with an onset between the ages of 14 and 40 years (average 19 years). Screening of
Page 5 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
6
the French cohort was performed by the direct sequencing of exon 7 in (1) Ist
Department of Pediatrics, Semmelweis University, Budapest, Hungary (2) INSERM
U983, Necker Hospital, Paris, France. The results from PodoNet consortium were
obtained through direct sequencing of the entire NPHS2 coding sequence performed
in the laboratories of the project partners.
Primers are available upon request. For sequence analysis, the software Sequencher
(Gene Codes, Ann Arbor, MI) was used. Segregation was confirmed by direct
sequencing of parental samples when available. All experiments were in accordance
with the Hungarian ethical committee recommendations and with the Declaration of
Helsinki Principles.
Haplotype analysis
Family members of patients carrying p.V290M mutation were genotyped using
markers D1S3760, D1S215, D1S3759 and D1S2751 neighboring the NPHS2 gene
locus. Genotyping was performed by PCR using fluorescent dye-labeled forward
primers and subsequent capillary electrophoresis using an 3500 Genetic Analyzer
(ABI), the results were analysed using GeneMapper software (Applied Biosystems).
Haplotype frequencies were compared using Fisher's exact test.
Page 6 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
7
Results
NPHS2 mutations
Seven of 38 unrelated patients (18%) were found to carry homozygous or compound
heterozygous NPHS2 mutations (Table 1). One of them, a nonsense mutation
(c.790G>T, p.E264*) was novel. While 5 patients carried a truncating or the similarly
severe p.R138Q mutation [24] on both alleles, 2 patients carried the p.V290M
mutation either in homozygous state (VM86) or in association to the p.R138Q
mutation (VM163) (Figure 1). In 3 patients (VM70, VM71 and VM84) only one allele
was found to be mutated carrying either a truncating, the p.V290M or the p.R138Q
mutation, respectively (Table 1).
Four patients carried the p.R229Q variant in heterozygous state. However, as no
pathogenic mutation was found on the second NPHS2 allele, p.R229Q was not
considered to be pathogenic in these patients.
Clinical phenotype of patients with NPHS2 mutations
The clinical presentation of the 5 patients with truncating or the p.R138Q mutation on
both alleles corresponded to the classical NPHS2-related phenotype [4] (Table 1). All
of them presented with nephrotic syndrome between the ages of 3 months and 4
years, and 4 of them progressed to ESRD between the ages of 2.5 and 9.5 years.
None of them responded to cyclosporin therapy and no recurrence was seen after
transplantation. While the phenotype of 2 patients (VM70, VM71) with a single
heterozygous mutation was similar (Table 1), patient VM84 achieved complete
remission (proteinuria<4mg/m2/hour) 8 weeks after the start of cyclosporine therapy.
Her proteinuria has been remaining in the normal range for 1,5 years, with no
medication in the last 6 months.
Page 7 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
8
In contrast to patients with truncating or p.R138Q mutations, the two adults carrying
the p.V290M mutation followed a markedly milder clinical course (Table 1). Patient
VM86 carrying the homozygous p.V290M mutation was diagnosed with proteinuria at
the age of 9.7 years through a school screening program. His proteinuria has been
persisted with an average value of 34mg/m2/hour (range: 3-104, n=119
measurements) (Figure 2). However, till now, the age of 18 years, he has never
developed edema and his serum albumin concentration dropped below 30g/l only
once (Figure 2). At the age of 16 years, a renal biopsy was performed which showed
FSGS. Currently, his GFR is estimated to be 108ml/min/1,73m2 by the MDRD
formula.
The second patient (VM163), being compound heterozygous for p.V290M and
p.R138Q, was diagnosed with proteinuria just before her 14th birthday through a
school screening program as well. Her proteinuria has been persisted with an
important variation (average: 35mg/m2/hour [range: 1-228, n=91]) (Figure 2). A renal
biopsy was performed at the age of 17.6 years which showed MCD. Her serum
albumin level first dropped below 30g/l at the age of 24.3 years, but had not
developed edema till the age of 27.5 years (Figure 2). She became pregnant at the
age of 28.3 years. At the 34th gestational week severe edema developed which
necessitated the termination of the pregnancy by cesarean section. The following
days, the edema resolved quickly. Currently, at the age of 31 years, her renal
function is still normal with an estimated (MDRD) GFR of 84 ml/min/1,73m2.
Page 8 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
9
Allele frequency of p.V290M in late-onset SRNS in the French and the PodoNet
cohorts
As the c.868G>A (p.V290M) mutation was associated with a late-onset SRNS in two
patients and its allele frequency was important (4/76 [5%] alleles) in the Hungarian
cohort, we examined its allele frequency in late-onset SRNS in two large European
cohorts. While none of the 83 patients in the French cohort, 2 out of 95 patients in the
PodoNet cohort was found to carry the p.V290M mutation, either in compound
heterozygous state in association to a splice site mutation (c.451+3A>T, intron 3) in a
German patient or as a single heterozygous mutation in a Turkish patient giving an
allele frequency of 1,1% in the PodoNet cohort.
Haplotype analysis of the p.V290M allele
To investigate whether a founder effect can explain the observed geographical
differences, the 12 parental alleles of the 3 patients carrying the p.V290M were
genotyped. Indeed, a common V290M haplotype was found with allele numbers
arbitrarily designated 8-3-1 based on (CA)n repeat number of 174 bp, 193 bp, and
180 bp at markers D1S3760, D1S215 and D1S3759 (Figure 3). The 8 non-V290M
alleles were all different from each other, and only one of them was haploidentical
with the V290M alleles within the conserved region of 530kb (Figure 3). Thus, the
marked difference in the frequency of this haplotype (4/4 vs. 1/8, p=0.01), supports
the notion that the p.V290M allele derives from a common ancestor.
Page 9 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
10
Discussion
Identification of a mutation in a patient with SRNS has direct clinical consequences, it
makes unnecessary further immunosuppressive treatment and unlikely the
recurrence of nephrotic syndrome in a kidney graft [25]. In the present study, the
finding of a single heterozygous NPHS2 mutation in a patient who achieved complete
remission after cyclosporine therapy does not contradict to this, instead it rather
emphasizes that the identification of an NPHS2 mutation carrier by chance is not
impossible in the immune form of SRNS. Therefore, despite of the general
expectation that the second mutation goes undetected; patients with one
heterozygous mutation should be treated as potential carriers in whom NPHS2 is not
pathogenic. Thus, considering only patients with mutations on both alleles, the
mutation rate of NPHS2 in isolated FSGS and MCD in the presented cohort (7/38
patients) corresponds to previous studies [17-19, 26].
Current guidelines propose the routine sequencing of NPHS2 only in childhood-onset
SRNS [4] or in patients with an onset before the age of 13 years [16]. This approach
is based on the observation that even an onset above the age of 10 years is rare and
has only been reported in patients with splice or missense mutations as p.V180M,
p.R238S and p.V290M [17-21]. Weber et al. found the p.V180M and the R238S
mutations in seven patients with a median age of onset of 11±1 years [18]. In a large
survey, among patients with two NPHS2 mutations, an onset above 10 years of age
was only found in five patients carrying either p.V180M or p.V290M [17]. The latest
onset associated with these mutations was 16.6 and 14.3 years, respectively.
Nevertheless, the mutations p.R238S and p.V180M – encoded by the same exon 5
as the p.R229Q variant – cannot be missed in adolescent-onset SRNS if the
Page 10 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
11
proposed screening of p.R229Q is performed by direct sequencing of the whole
exon.
In contrast, the mutation p.V290M – reported to lead to SRNS between the ages of 5
months and 14.7 years in 10 patients [17, 19, 27-30] – is encoded by exon 7. The
proposed sequencing of all NPHS2 exons till the age of onset of 14 years, could
identify this mutation in all but one case. In this study, we report two patients carrying
p.V290M mutation with an unexpectedly late onset and mild clinical course. The 18-
year-old boy has never had edema and his serum albumin level is still in the normal
range. The 31-year-old woman developed first edema at the age of 27.5 years, and
currently has a normal renal function. As patients with two NPHS2 mutations have
not been reported to progress to ESRD later than 26 years of age [17-20, 22], this
clinical course is unexpectedly mild and comparable to that of patients carrying
p.R229Q in trans-association to a mutation. Along the same line, 2 patients with
p.V290M were reported to progress to ESRD, at the ages of 20 and 24 years [29,
30]. In the current study, both patients carrying p.V290M had been diagnosed with
proteinuria by school screening programs. However, based on the age at the onset of
clinical symptoms and the current guidelines, only the p.R229Q variant should have
been screened, and thus the mutation screening would have failed.
The clinical significance of this observation depends on the geographic region. While
the allele frequency of the p.V290M mutation was found to be 1/13.000 in the general
American population [31], it has never been reported in SRNS patients from Western
Europe or North Africa [13, 18, 22]; but was found in 11 of 124 patients with two
NPHS2 mutations from Central and Eastern European cohorts, including the present
one [17, 19, 25, 27, 28], suggesting a founder effect. Accordingly, in the present
study, none of the French or Italian patients with adolescent- and adult-onset SRNS,
Page 11 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
12
but 2 of 46 patients from Germany, Poland or Turkey carried this mutation. The
founder effect of the p.V290M mutation was further supported by the finding that the
four V290M alleles found in the present study were haploidentical in an at least
0.5Mb region.
Since the original descriptions, the spectrum of SRNS phenotypes linked to a single
gene has been continuously expanding. Mutations of NPHS1 were found in
childhood- and adult-onset SRNS [32, 33], as such as NPHS2 mutations in neonatal-
onset SRNS [18]. It has been pointed out that most of this phenotypic variability is
secondary to the mutations’ different severity and effect on the encoded protein’s
function [17]. As the size of the cohorts and the clinical experience are growing, the
mutation screening can be expected to become more specific based on subtle
phenotype-differences. Along this line, while the mutation screening of NPHS2 in
adolescent and adult-onset SRNS was focused on p.R229Q [4, 16], recently,
sequencing of the entire coding region of NPHS2 in patients with an onset of SRNS
below 20 years of age has been recently proposed by the PodoNet cohort [34].
In conclusion, based on the presented observations, we are going to screen patients
with an onset of SRNS below 30 years not only for the p.R229Q variant but also for
the p.V290M mutation. We propose that this missense mutation should be screened
in young adults with SRNS from Central and Eastern Europe.
Page 12 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
13
Acknowledgement
This work was supported by Pfizer, TÁMOP-4.2.1/B-09/1/KMR-2010-0001, Zoltán
Magyary and János Bólyai fellowship (KT). The authors thank Mária Bernáth for
expert technical assistance.
References
1. Mekahli D, Liutkus A, Ranchin B, Yu A, Bessenay L, Girardin E, Van Damme-Lombaerts R,
Palcoux JB, Cachat F, Lavocat MP, Bourdat-Michel G, Nobili F, Cochat P (2009) Long-term
outcome of idiopathic steroid-resistant nephrotic syndrome: a multicenter study. Pediatr
Nephrol 24:1525-1532.
2. (1981) The primary nephrotic syndrome in children. Identification of patients with minimal
change nephrotic syndrome from initial response to prednisone. A report of the International
Study of Kidney Disease in Children. J Pediatr 98:561-564.
3. Tune BM, Mendoza SA (1997) Treatment of the idiopathic nephrotic syndrome: regimens and
outcomes in children and adults. J Am Soc Nephrol 8:824-832.
4. Benoit G, Machuca E, Antignac C (2010) Hereditary nephrotic syndrome: a systematic
approach for genetic testing and a review of associated podocyte gene mutations. Pediatr
Nephrol 25:1621-1632.
5. Ozaltin F, Ibsirlioglu T, Taskiran EZ, Baydar DE, Kaymaz F, Buyukcelik M, Kilic BD, Balat A,
Iatropoulos P, Asan E, Akarsu NA, Schaefer F, Yilmaz E, Bakkaloglu A (2011) Disruption of
PTPRO causes childhood-onset nephrotic syndrome. Am J Hum Genet 89:139-147.
6. Brown EJ, Schlondorff JS, Becker DJ, Tsukaguchi H, Tonna SJ, Uscinski AL, Higgs HN,
Henderson JM, Pollak MR (2010) Mutations in the formin gene INF2 cause focal segmental
glomerulosclerosis. Nat Genet 42:72-76.
7. Kaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong HQ, Mathis BJ, Rodriguez-Perez JC,
Allen PG, Beggs AH, Pollak MR (2000) Mutations in ACTN4, encoding alpha-actinin-4, cause
familial focal segmental glomerulosclerosis. Nat Genet 24:251-256.
8. Matejas V, Hinkes B, Alkandari F, Al-Gazali L, Annexstad E, Aytac MB, Barrow M, Blahova K,
Bockenhauer D, Cheong HI, Maruniak-Chudek I, Cochat P, Dotsch J, Gajjar P, Hennekam RC,
Janssen F, Kagan M, Kariminejad A, Kemper MJ, Koenig J, Kogan J, Kroes HY, Kuwertz-Broking
E, Lewanda AF, Medeira A, Muscheites J, Niaudet P, Pierson M, Saggar A, Seaver L, Suri M,
Tsygin A, Wuhl E, Zurowska A, Uebe S, Hildebrandt F, Antignac C, Zenker M (2010) Mutations
in the human laminin beta2 (LAMB2) gene and the associated phenotypic spectrum. Hum
Mutat 31:992-1002.
9. Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, Hawkins AF, Daskalakis N, Kwan SY,
Ebersviller S, Burchette JL, Pericak-Vance MA, Howell DN, Vance JM, Rosenberg PB (2005) A
mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis.
Science 308:1801-1804.
10. Lowik MM, Groenen PJ, Pronk I, Lilien MR, Goldschmeding R, Dijkman HB, Levtchenko EN,
Monnens LA, van den Heuvel LP (2007) Focal segmental glomerulosclerosis in a patient
homozygous for a CD2AP mutation. Kidney Int 72:1198-1203.
11. Hinkes B, Wiggins RC, Gbadegesin R, Vlangos CN, Seelow D, Nurnberg G, Garg P, Verma R,
Chaib H, Hoskins BE, Ashraf S, Becker C, Hennies HC, Goyal M, Wharram BL, Schachter AD,
Page 13 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
14
Mudumana S, Drummond I, Kerjaschki D, Waldherr R, Dietrich A, Ozaltin F, Bakkaloglu A,
Cleper R, Basel-Vanagaite L, Pohl M, Griebel M, Tsygin AN, Soylu A, Muller D, Sorli CS, Bunney
TD, Katan M, Liu J, Attanasio M, O'Toole J F, Hasselbacher K, Mucha B, Otto EA, Airik R,
Kispert A, Kelley GG, Smrcka AV, Gudermann T, Holzman LB, Nurnberg P, Hildebrandt F
(2006) Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome
variant that may be reversible. Nat Genet 38:1397-1405.
12. Mucha B, Ozaltin F, Hinkes BG, Hasselbacher K, Ruf RG, Schultheiss M, Hangan D, Hoskins BE,
Everding AS, Bogdanovic R, Seeman T, Hoppe B, Hildebrandt F (2006) Mutations in the
Wilms' tumor 1 gene cause isolated steroid resistant nephrotic syndrome and occur in exons
8 and 9. Pediatr Res 59:325-331.
13. Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler MC,
Niaudet P, Antignac C (2000) NPHS2, encoding the glomerular protein podocin, is mutated in
autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet 24:349-354.
14. Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V,
Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K
(1998) Positionally cloned gene for a novel glomerular protein--nephrin--is mutated in
congenital nephrotic syndrome. Mol Cell 1:575-582.
15. Hinkes BG, Mucha B, Vlangos CN, Gbadegesin R, Liu J, Hasselbacher K, Hangan D, Ozaltin F,
Zenker M, Hildebrandt F (2007) Nephrotic syndrome in the first year of life: two thirds of
cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2). Pediatrics
119:e907-919.
16. Santin S, Bullich G, Tazon-Vega B, Garcia-Maset R, Gimenez I, Silva I, Ruiz P, Ballarin J, Torra
R, Ars E (2011) Clinical utility of genetic testing in children and adults with steroid-resistant
nephrotic syndrome. Clin J Am Soc Nephrol 6:1139-1148.
17. Hinkes B, Vlangos C, Heeringa S, Mucha B, Gbadegesin R, Liu J, Hasselbacher K, Ozaltin F,
Hildebrandt F (2008) Specific podocin mutations correlate with age of onset in steroid-
resistant nephrotic syndrome. J Am Soc Nephrol 19:365-371.
18. Weber S, Gribouval O, Esquivel EL, Moriniere V, Tete MJ, Legendre C, Niaudet P, Antignac C
(2004) NPHS2 mutation analysis shows genetic heterogeneity of steroid-resistant nephrotic
syndrome and low post-transplant recurrence. Kidney Int 66:571-579.
19. Berdeli A, Mir S, Yavascan O, Serdaroglu E, Bak M, Aksu N, Oner A, Anarat A, Donmez O, Yildiz
N, Sever L, Tabel Y, Dusunsel R, Sonmez F, Cakar N (2007) NPHS2 (podicin) mutations in
Turkish children with idiopathic nephrotic syndrome. Pediatr Nephrol 22:2031-2040.
20. Bower M, Salomon R, Allanson J, Antignac C, Benedicenti F, Benetti E, Binenbaum G, Jensen
UB, Cochat P, DeCramer S, Dixon J, Drouin R, Falk MJ, Feret H, Gise R, Hunter A, Johnson K,
Kumar R, Lavocat MP, Martin L, Moriniere V, Mowat D, Murer L, Nguyen HT, Peretz-Amit G,
Pierce E, Place E, Rodig N, Salerno A, Sastry S, Sato T, Sayer JA, Schaafsma GC, Shoemaker L,
Stockton DW, Tan WH, Tenconi R, Vanhille P, Vats A, Wang X, Warman B, Weleber RG, White
SM, Wilson-Brackett C, Zand DJ, Eccles M, Schimmenti LA, Heidet L (2012) Update of PAX2
mutations in renal coloboma syndrome and establishment of a locus-specific database. Hum
Mutat 33:457-466.
21. Hildebrandt F, Heeringa SF (2009) Specific podocin mutations determine age of onset of
nephrotic syndrome all the way into adult life. Kidney Int 75:669-671.
22. Machuca E, Hummel A, Nevo F, Dantal J, Martinez F, Al-Sabban E, Baudouin V, Abel L,
Grunfeld JP, Antignac C (2009) Clinical and epidemiological assessment of steroid-resistant
nephrotic syndrome associated with the NPHS2 R229Q variant. Kidney Int 75:727-735.
23. Tsukaguchi H, Sudhakar A, Le TC, Nguyen T, Yao J, Schwimmer JA, Schachter AD, Poch E,
Abreu PF, Appel GB, Pereira AB, Kalluri R, Pollak MR (2002) NPHS2 mutations in late-onset
focal segmental glomerulosclerosis: R229Q is a common disease-associated allele. J Clin
Invest 110:1659-1666.
24. Huber TB, Simons M, Hartleben B, Sernetz L, Schmidts M, Gundlach E, Saleem MA, Walz G,
Benzing T (2003) Molecular basis of the functional podocin-nephrin complex: mutations in
Page 14 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
15
the NPHS2 gene disrupt nephrin targeting to lipid raft microdomains. Hum Mol Genet
12:3397-3405.
25. Buscher AK, Kranz B, Buscher R, Hildebrandt F, Dworniczak B, Pennekamp P, Kuwertz-Broking
E, Wingen AM, John U, Kemper M, Monnens L, Hoyer PF, Weber S, Konrad M (2010)
Immunosuppression and renal outcome in congenital and pediatric steroid-resistant
nephrotic syndrome. Clin J Am Soc Nephrol 5:2075-2084.
26. Caridi G, Bertelli R, Di Duca M, Dagnino M, Emma F, Onetti Muda A, Scolari F, Miglietti N,
Mazzucco G, Murer L, Carrea A, Massella L, Rizzoni G, Perfumo F, Ghiggeri GM (2003)
Broadening the spectrum of diseases related to podocin mutations. J Am Soc Nephrol
14:1278-1286.
27. Karle SM, Uetz B, Ronner V, Glaeser L, Hildebrandt F, Fuchshuber A (2002) Novel mutations
in NPHS2 detected in both familial and sporadic steroid-resistant nephrotic syndrome. J Am
Soc Nephrol 13:388-393.
28. Ruf RG, Lichtenberger A, Karle SM, Haas JP, Anacleto FE, Schultheiss M, Zalewski I, Imm A,
Ruf EM, Mucha B, Bagga A, Neuhaus T, Fuchshuber A, Bakkaloglu A, Hildebrandt F (2004)
Patients with mutations in NPHS2 (podocin) do not respond to standard steroid treatment of
nephrotic syndrome. J Am Soc Nephrol 15:722-732.
29. Skalova S, Podhola M, Vondrak K, Chernin G (2010) Plasmapheresis-induced clinical
improvement in a patient with steroid-resistant nephrotic syndrome due to podocin (NPHS2)
gene mutation. Acta Medica (Hradec Kralove) 53:157-159.
30. Reiterova J, Safrankova H, Obeidova L, Stekrova J, Maixnerova D, Merta M, Tesar V (2012)
Mutational Analysis of the NPHS2 Gene in Czech Patients with Idiopathic Nephrotic
Syndrome. Folia biologica 58:64-68.
31. Exome Variant Server NGESPE Seattle, WA (URL: http://evs.gs.washington.edu/EVS/).
32. Philippe A, Nevo F, Esquivel EL, Reklaityte D, Gribouval O, Tete MJ, Loirat C, Dantal J,
Fischbach M, Pouteil-Noble C, Decramer S, Hoehne M, Benzing T, Charbit M, Niaudet P,
Antignac C (2008) Nephrin mutations can cause childhood-onset steroid-resistant nephrotic
syndrome. J Am Soc Nephrol 19:1871-1878.
33. Santin S, Garcia-Maset R, Ruiz P, Gimenez I, Zamora I, Pena A, Madrid A, Camacho JA, Fraga
G, Sanchez-Moreno A, Cobo MA, Bernis C, Ortiz A, de Pablos AL, Pintos G, Justa ML, Hidalgo-
Barquero E, Fernandez-Llama P, Ballarin J, Ars E, Torra R (2009) Nephrin mutations cause
childhood- and adult-onset focal segmental glomerulosclerosis. Kidney Int 76:1268-1276.
34. Lipska B, Trautmann, A., Anarat, A., Ozaltin, F., Caridi, G., Noris, M., Schaefer, F, P.
Consortium (2011) The role of NPHS2 and INF2 in adolescent-onset steroid-resistant
nephrotic syndrome (SRNS): finding in the PodoNet cohort 26:1577.
Page 15 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
16
Legends to figures
Figure 1
Sequencing chromatogram of the c.868 G>A (p.V290M) mutation in heterozygous
(patient VM163) and homozygous state (patient VM86)
Figure 2
Proteinuria and serum albumin level of patients VM86 and VM163 carrying p.V290M
mutation
Figure 3
Parental haplotypes of patients carrying the p.V290M mutation (P: paternal, M:
maternal alleles). The common parts of the four V290M alleles are highlighted (gray).
Only one of the 8 non-V290M alleles is haploidentical with them (outlined by a
dashed line). All the non-V290M alleles are different from each other.
Page 16 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
Table 1. Genetic and clinical characteristics of patients with NPHS2 mutations
Ind Gender Nucleotide change Amino acid
change Histology
Age at dg of
proteinuria (years)
Age at onset of
edema (years)
Response to
cyclosporin
Age at ESRD
(years)
Recurrence after
Tx
Patients carrying truncating or p.R138Q mutations on both alleles
VM1 F c.413G>A het (m)
c.948delT het (p)
p.R138Q
p.A317Lfs*31 FSGS 4 4 resistent N (7.5) no Tx
VM83 M c.413G>A het (p)
c.790G>T het (m)
p.R138Q
p.E264* FSGS 4 4 resistent ESRD (9.5) no recurrence
VM161 M c.413G>A hom p.R138Q FSGS 0.3 0.3 resistent ESRD (6) no recurrence
VM162 F c.413G>A het
c.419delG het
p.R138Q
p.G140Dfs*41 FSGS 1.5 1.5 resistent ESRD (6) no recurrence
VM164 F c.467dupT het
c.948delT het
p.L156Ffs*11
p.A317Lfs*31 FSGS 3 3 resistent ESRD (9) no recurrence
Patients carrying p.V290M mutation in homozygous or compound heterozygous state
VM86 M c.868G>A hom (m, p) p.V290M FSGS 9 not yet (18) resistent N (18) no Tx
VM163 F c.413G>A het (m)
c.868G>A het (p)
p.R138Q
p.V290M MCD 14 27.5 resistent N (31) no Tx
Patients with a single heterozygous mutation
VM70 M c.948delT het p.A317Lfs*31 FSGS 0.7 0.7 resistent ESRD (2.5) no recurrence
VM71 F c.868G>A het p.V290M FSGS 3 6 not treated ESRD (8) no recurrence
VM84 F c.413G>A het p.R138Q FSGS 5 5 complete
remission N (6.5) no Tx
dg: diagnosis, FSGS: focal segmental glomerulosclerosis, het: heterozygous, hom: homozygous, m: maternal allele, MCD: minimal change disease,
N: normal renal function, p: paternal allele, Tx: renal transplantation
Page 33 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
Sequencing chromatogram of the c.868 G>A (p.V290M) mutation in heterozygous (patient VM163) and homozygous state (patient VM86)
23x27mm (300 x 300 DPI)
Page 34 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
Proteinuria and serum albumin level of patients VM86 and VM163 carrying p.V290M mutation 82x123mm (300 x 300 DPI)
Page 35 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
Parental haplotypes of patients carrying the p.V290M mutation (P: paternal, M: maternal alleles). The common parts of the four V290M alleles are highlighted (gray). Only one of the 8 non-V290M alleles is haploidentical with them (outlined by a dashed line). All the non-V290M alleles are different from each
other.
Page 36 of 36
Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]
Pediatric Nephrology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
Top Related